Our long-term goal is to determine the role(s) of free radicals and their derivatives in regulating contractile function of limb and respiratory skeletal muscle. In Years 4-7, we propose a comprehensive investigation of NO biology in muscle that has four Specific Aims: Aim 1. To evaluate NO production and its regulation in skeletal muscle. We postulate that NO is produced by type II muscle fibers (Hypothesis 1A) and will test this by localizing NOS among fiber populations of individual muscles and by measuring NO production in muscles with different fiber compositions. We also will test the postulate that NO production is increased by stimulating muscle to contract (Hypothesis 1B). Aim 2. To determine NO effects on skeletal muscle function. Our data suggest that NO inhibits contractile function of unfatigued skeletal muscle (Hypothesis 2A). We will evaluate this in unfatigued muscle. Preliminary data further suggest that NO inhibits acute muscular fatigue (Hypothesis 2B) which we propose to test by altering NO levels during fatiguing contractions. Aim 3. To evaluate cGMP as a second messenger for NO effects. We hypothesize that cGMP depresses skeletal muscle function (Hypothesis 3A) and will test this by measuring contractile changes caused by perturbing cGMP metabolism. We also postulate that NO regulates tissue cGMP levels (Hypothesis 3B) and will test this by localizing cGMP in the tissue and by measuring NO effects on cGMP. Aim 4. To evaluate intracellular mechanism(s) of NO effects. Contractile studies suggest that calcium release may be inhibited by NO (Hypothesis 4A). We will test free radical effects on calcium release by measuring force production and calcium transients in single fibers isolated from mouse limb muscle. We also will test the hypothesis that myofilament interaction is inhibited by NO (Hypothesis 4B) by evaluating mechanically-skinned single fibers for NO effects on the calcium-force and force-velocity relationships.